Polymerization-depolymerization Cycles Research Articles

Designing Thermally Stable Organocatalysts for Poly(ethylene terephthalate) Synthesis: Toward a One-Pot, Closed-Loop Chemical Recycling System for PET

Organocatalysis provides robust methodology to furnish “greener” routes to polymer synthesis. However, the application toward the synthesis of aromatic polymers via step-growth polymerization is an area that justifies more investigation, as a consequence of the poor thermal stability of many organic catalysts and the high reaction temperatures commonly required. In this study, thermally stable organic salts consisting of an organic base and an organic acid were explored to understand key elements required for the bulk synthesis of poly(ethylene terephthalate) (PET) at 270 °C. The ΔpKa values of the salts played an important role in the thermal stability such that the salts with higher ΔpKa values showed higher stability because of the strong acid–base interactions. The 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) salts with high ΔpKa values (≥16.9) showed the best catalytic activity among the investigated salts in terms of both low amounts of side reactions and discoloration. The thermal and chemical stability of the salts also affected the polymer properties. Dimerization side reactions that lead to defects in the polymer backbone were found to occur more readily in salts containing strong acids as components, particularly as the ΔpKa between the acid–base components decreased. The discoloration of the PET sample was also correlated to the thermal stability of the organic salt catalyst, with a lower stability generally leading to enhanced discoloration likely due to decomposition of base components. Polymerization–depolymerization cycles were also investigated with the TBD:p-toluenesulfonic acid (TSA) salt and the feasibility of simple, closed-loop recycling of PET with the system was established.

Control of actin polymerization via reactive oxygen species generation using light or radiation.

Actin is one of the most prevalent proteins in cells, and its amino acid sequence is remarkably conserved from protozoa to humans. The polymerization-depolymerization cycle of actin immediately below the plasma membrane regulates cell function, motility, and morphology. It is known that actin and other actin-binding proteins are targets for reactive oxygen species (ROS), indicating that ROS affects cells through actin reorganization. Several researchers have attempted to control actin polymerization from outside the cell to mimic or inhibit actin reorganization. To modify the polymerization state of actin, ultraviolet, visible, and near-infrared light, ionizing radiation, and chromophore-assisted light inactivation have all been reported to induce ROS. Additionally, a combination of the fluorescent protein KillerRed and the luminescent protein luciferase can generate ROS on actin fibers and promote actin polymerization. These techniques are very useful tools for analyzing the relationship between ROS and cell function, movement, and morphology, and are also expected to be used in therapeutics. In this mini review, we offer an overview of the advancements in this field, with a particular focus on how to control intracellular actin polymerization using such optical approaches, and discuss future challenges.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.

Autonomous helical propagation of active toroids with mechanical action

Self-assembly in nature is fundamentally dynamic, existing in out-of-equilibrium state in which the systems have the ability to autonomously respond to environmental changes. However, artificial systems exist in a global minimum state, which are incapable of conducting such complex functions. Here we report that input of thermal energy can trigger fixed, artificial toroids to spontaneously nucleate helical growth. The helical polymerization undergoes reversible and repeatable cycles with subsequent energy input. When the toroids are located inside lipid vesicles, the polymerization-depolymerization cycle is accompanied by reversible elongation of spherical vesicles. Such liberation from a global minimum state will pave the way to create emergent structures with functions as complex as those of living systems.

Non-Straub type actin from molluscan catch muscle

We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Сrenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for the extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization-depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers.

Role of the Actin Ala-108–Pro-112 Loop in Actin Polymerization and ATPase Activities

Actin plays fundamental roles in a variety of cell functions in eukaryotic cells. The polymerization-depolymerization cycle, between monomeric G-actin and fibrous F-actin, drives essential cell processes. Recently, we proposed the atomic model for the F-actin structure and found that actin was in the twisted form in the monomer and in the untwisted form in the filament. To understand how the polymerization process is regulated (Caspar, D. L. (1991) Curr. Biol. 1, 30-32), we need to know further details about the transition from the twisted to the untwisted form. For this purpose, we focused our attention on the Ala-108-Pro-112 loop, which must play crucial roles in the transition, and analyzed the consequences of the amino acid replacements on the polymerization process. As compared with the wild type, the polymerization of P109A was accelerated in both the nucleation and the elongation steps, and this was attributed to an increase in the frequency factor of the Arrhenius equation. The multiple conformations allowed by the substitution presumably resulted in the effective formation of the collision complex, thus accelerating polymerization. On the other hand, the A108G mutation reduced the rates of both nucleation and elongation due to an increase in the activation energy. In the cases of polymerization acceleration and deceleration, each functional aberration is attributed to a distinct elementary process. The rigidity of the loop, which mediates neither too strong nor too weak interactions between subdomains 1 and 3, might play crucial roles in actin polymerization.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Two Drosophila Myosin Transducer Mutants with Distinct Cardiomyopathies Have Divergent ADP and Actin Affinities

Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila.

The Putative RNA-Processing Protein, THO2, is a Microtubule-Associated Protein in Tobacco

THO2 is a component of the THO-TREX (transcription and export factor) complex that participates in mRNA metabolism and export from the nucleus in yeast and animal cells. Here we report that tobacco putative THO2-related protein (NtTHO2) is a microtubule-associated protein, which directly binds to microtubules in vitro and co-localizes with cortical microtubules in vivo. We purified endogenous NtTHO2 by cycles of microtubule polymerization-depolymerization from crude extracts of tobacco BY-2 miniprotoplasts. Purified NtTHO2 sedimented with microtubules in vitro. Immunofluorescence revealed that NtTHO2 was present in both the nucleus and cytoplasm. In interphase, cytoplasmic NtTHO2 was localized along cortical microtubules. In the mitotic phase, NtTHO2 was localized to the mitotic spindle but not to either the preprophase band or the phragmoplast. In mature cells of seedling roots, and in BY-2 cells in which proliferation was stopped by removing 2,4-D, NtTHO2 staining was confined mainly to the nucleolus. These results suggest that NtTHO2 is a multifunctional protein that participates in mRNA metabolism, and also functions within the cortical microtubules and mitotic spindle.

Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

TTLL7 Is a Mammalian β-Tubulin Polyglutamylase Required for Growth of MAP2-positive Neurites

Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for alpha-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.

Microtubule cytoskeleton involvement in muscarinic suppression of voltage-gated calcium channel current in guinea-pig ileal smooth muscle.

1. Effects of agents, which affect microtubule polymerization-depolymerization cycle, on Ba2+ current (IBa) flowing through voltage-gated Ca2+ channels and carbachol (CCh)-induced sustained suppression of IBa were examined in whole-cell voltage-clamped smooth muscle cells of guinea-pig ileum. 2. offchicine (100 microM) and vinblastine (100 microM), microtubule depolymerizers, increased the ampitude of IBa. Lumicolchicine (100 microM), an inactive analogue of colchicine, had no effect on IBa. 3. Taxol (1 - 100 microM), a microtubule polymerizer, decreased IBa in a concentration-dependent manner and accelerated the rate of inactivation of IBa. Baccatin III (100 microM), an inactive analogue of taxol, had no effect on IBa. 4. Colchicine (100 microM) and vinblastine (100 microM), but not lumicolchicine (100 microM), decreased or abolished the sustained component of CCh (10 microM)-induced IBa suppression. 5. Pretreatment with taxol (10 - 100 microM) resulted in a concentration-dependent decrease in IBa and the action of CCh on IBa. The inhibitory effects of taxol and CCh on IBa were not additive. 6. Colchicine (100 microM) or taxol (100 microM) had no effect on voltage-gated K+ channel current or CCh-induced non-selective cationic channel current. 7. These results suggest that polymerization of microtubules leads to suppression of Ca2+ channel activity, and that muscarinic sustained suppression of Ca2+ channel current is mediated by a signal transduction element which involves microtubule cytoskeleton.

Immediate disruption of spindle poles and induction of additional microtubule-organizing centres by a phenylcarbamate, during plant mitosis

The herbicide carbetamide [(R)-1-(ethylcarbamoyl) ethylphenylcarbamate], in the 0.4 to 0.8 mM range, efficiently induced multipolar mitoses inAllium cepa L. The frequency of multipolar anaphases rose earlier and reached higher values when both concentration and time of treatment increased, up to a maximum of 90% after 1 h of treatment. To identify the physiological target, the kinetics of induction of multipolar mitoses were followed during recovery from very short treatments (5, 10, and 15 min). Tubulin immunodetection showed that phenylcarbamate immediately disrupts the cohesion between the different bundles of microtubule minus ends which converge at the pole. The spindle was rendered multipolar about three times more efficiently in metaphase than in anaphase. The observations do not support any effect of the herbicide on the tubulin polymerization-depolymerization cycle, and suggest that the minus ends of the microtubules remained stabilized in carbetamide. Thus, the density of kinetochore microtubules and their lengths were unmodified in the individual chromosomes which became detached from both spindle poles in response to the herbicide. Extra microtubule-organizing centres for the assembly of both preprophase band and phragmoplast (the tubulin arrays which characterize the microtubular cycle responsible for cytokinesis in plant cells) were also rapidly induced.

Modeling Microtubule Oscillations

Synchronization of molecular reactions in a macroscopic volume may cause the volume's physical properties to change dynamically and thus reveal much about the reactions. As an example, experimental time series for so-called microtubule oscillations are analyzed in terms of a minimal model for this complex polymerization-depolymerization cycle. The model reproduces well the qualitatively different time series that result from different experimental conditions, and illuminates the role and importance of individual processes in the cycle. Simple experiments are suggested that can further test and define the model and the polymer's reaction cycle.

Thermally Controlled Formation of Fullerene−Diene Oligomers and Copolymers

A fullerene-based polymer has been obtained by the Diels-Alder cycloaddition of C60 with the bis-anthracene monomer Anth-CH 2 O(CH 2 ) 10 OCH 2 -Anth. This addition is fully reversible upon heating to 75°C, providing a fully thermoreversible polymer capable of multiple polymerization-depolymerization cycles

"Varicoid change" of bile canaliculi in rat liver at an early phase of ischaemia-reperfusion injury.

To elucidate early changes and the mechanism of ischaemia-reperfusion liver injury, we investigated three-dimensional microstructural changes of cellular actin filaments in rat livers using confocal laser scanning microscopy. The liver tissues of a reperfusion group were examined 12 h after removal of a vascular clamp. Fixed tissues were stained with fluorescein-labelled phalloidin to obtain stereoscopic images of the actin filaments and these were compared with histological findings. The images of bile canaliculi showed that multiple abnormal minute diverticula arose from the canalicular membranes and fused with one another, resulting in irregular dilation of the bile canaliculi. These changes were observed after 15 min of ischaemia and reperfusion in which no significant necrosis was seen. The frequency and degree of these changes were strictly dependent on the periods of ischaemia (15-60 min). We called these bile canacilular lesions "varicoid changes". The liver of an ischaemia group taken after persistent clamping without reperfusion did not show these changes. Our findings suggest that the varicoid change in the bile canaliculi is probably due to alterations in the actin polymerization-depolymerization cycle and is a pathognomonic change of ischaemia-reperfusion liver injury.

Poliovirus infection results in structural alteration of a microtubule-associated protein.

Poliovirus infection results in profound changes in cellular metabolism and architecture. To identify alterations in cellular proteins following poliovirus infection which might account for these changes, monoclonal antibodies were prepared by screening for differences in antigen pattern in infected and uninfected cell lysates. Further characterization of the antigen of one such antibody (25 C C1) is described in this report. The 25 C C1 antigen is a cytoskeleton-associated protein which decreases in size 4 to 5 h postinfection. It copurifies with some of the protein synthesis initiation factors but not with eucaryotic initiation factor (eIF)-4F, the p220 subunit of which is cleaved following infection (D. Etchison, S. C. Milburn, I. Edery, N. Sonenberg, and J. W. B. Hershey, J. Biol. Chem. 257:14806-14810, 1982). Unlike alteration of p220, alteration of the 25 C C1 antigen is not due to a protease which can be detected by cell lysate mixing experiments. Alteration of the antigen occurs during purification, suggesting progressive proteolysis, but the alteration is more extensive in preparations from infected cells than in those from uninfected cells. A recombinant phage expressing the antigenic determinant was isolated from a human fibroblast cDNA library, and the sequence of the cDNA insert was found to be entirely contained within the established sequence of microtubule-associated protein (MAP) 4 (R. R. West, K. M. Tenbarge, and J. B. Olmsted, J. Biol. Chem. 266:21886-21896, 1991). The antigen distribution, as detected by indirect immunofluorescence, was similar to, but more diffuse than, the distribution of tubulin. The antibody recognized the largest abundant HeLa cell MAP, which copurified with tubulin after three cycles of polymerization-depolymerization, thus confirming the identity of the antigen as MAP 4. These results indicate that poliovirus infection of HeLa cells affects the structural integrity of a cytoskeletal protein, MAP 4.

Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: Evidence for an inhibitor of vincristine binding

In cytosols from human rhabdomyosarcoma xenografts, the formation of vincristine (VCR)-tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Biophys. Res. Commun., 135:695-700, 1986). We have further examined this modulation to determine whether a) GTP was protecting the VCR binding site from denaturation, b) the enhancement of complex formation was guanosine specific, and c) whether this influence was a direct interaction between GTP, VCR, and tubulin, or was mediated through another factor. In GTP-depleted cytosols from tumor xenografts HxRH18 and HxRh12, VCR binding activity was stable for at least 2 hours at 37 degrees C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 nM GTP increased complex formation slightly, with complex formation increasing as GTP concentrations were increased to 5 microM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation three-fold, while GMP, GMP-PNP, and ITP increased formation 1.5-fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific for the guanine nucleotides GDP and GTP. Microtubule protein, purified from Rh18 and Rh12 tumors by cycles of polymerization-depolymerization, bound VCR rapidly and binding was not influenced by GTP. This suggested that GTP modulation of VCR binding in cytosols was through a soluble factor lost in tubulin purification. In experiments with cytosol fractionated by molecular weight, there was inhibition of VCR binding activity by fractions with an mw range 20-50 kD. This inhibition was decreased by 25% by the addition of GTP. These data suggest that in tumor cytosols there may be competition between VCR and a natural ligand that is modulated by GTP. Two potential models for VCR binding are proposed.